Generation of 3D ex vivo mouse- and patient-derived glioma explant slice model for integration of confocal time-lapse imaging and spatial analysis.

Development of spatial-integrative pre-clinical models is needed for glioblastoma, which are heterogenous tumors with poor prognosis. Here, we present an optimized protocol to generate three-dimensional ex vivo explant slice glioma model from orthotopic tumors, genetically engineered mouse models, and fresh patient-derived specimens. We describe a step-by-step workflow for tissue acquisition, dissection, and sectioning of 300-μm tumor slices maintaining cell viability. The explant slice model allows the integration of confocal time-lapse imaging with spatial analysis for studying migration, invasion, and tumor microenvironment, making it a valuable platform for testing effective treatment modalities. For complete details on the use and execution of this protocol, please refer to Comba et al. (2022).1.